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J Hacker

Publications and source records attributed to J Hacker.

At least 199 records · Page 11Linked to original sources
PubMed1988

Gene clusters for S fimbrial adhesin (sfa) and F1C fimbriae (foc) of Escherichia coli: comparative aspects of structure and function.

Fimbrial adhesins enable bacteria to attach to eucaryotic cells. The genetic determinants for S fimbrial adhesins (sfa) and for F1C ("pseudotype I") fimbriae (foc) were compared. Sfa and F1C represent functionally distinct adhesins in their receptor specificities. Nevertheless, a high degree of homology between both determinants was found on the basis of DNA-DNA hybridizations. Characteristic differences in the restriction maps of the corresponding gene clusters, however, were visible in regions coding for the fimbrial subunits and for the S-specific adhesin. While a plasmid carrying the genetic determinant for F1C fimbriae was able to complement transposon-induced sfa mutants, a plasmid carrying the genetic determinant for a third adhesin type, termed P fimbriae, was unable to do so. Proximal sfa-specific sequences carrying the S fimbrial structural gene were fused to sequences representing the distal part of the foc gene cluster to form a hybrid cluster, and the foc proximal region coding for the structural protein was ligated to sfa distal sequences to form a second hybrid. Both hybrid clones produced intact fimbriae. Anti-F1C monoclonal antibodies (MAbs) only recognized clones which produced F1C fimbriae, and an anti-S adhesin MAb marked clones which expressed the S adhesin. However, one of four other anti-S fimbriae-specific MAbs reacted with both fimbrial structures, S and F1C, indicating a common epitope on both antigens. The results presented here support the view that sfa and foc determinants code for fimbriae that are similar in several aspects, while the P fimbriae are members of a more distantly related group.

Adhesins, Escherichia coli↗
PubMed1988

Binding sites in the rat brain for Escherichia coli S fimbriae associated with neonatal meningitis.

Escherichia coli strains that cause sepsis and meningitis in neonatal infants carry S fimbriae that bind to sialyl galactoside units of cell surface glycoproteins. To investigate the possible role of S fimbriae in determining the tissue tropism of neonatal meningitis, we have studied the presence of binding sites for S fimbriae in different tissues of the neonatal rat which is susceptible to meningitis caused by S-fimbriated E. coli. Purified S fimbriae were incubated on cryostat sections of different rat organs and their binding was assessed by indirect immunofluorescence. In the brain of the neonatal rat, S fimbriae specifically bound to the luminal surfaces of the vascular endothelium and of the epithelium lining the choroid plexuses and brain ventricles. The binding was completely inhibited by the trisaccharide NeuAc alpha 2-3Gal beta 1-4Glc, a receptor analogue of S fimbriae, and by a preceding neuraminidase treatment of the sections. A recombinant E. coli strain expressing S fimbriae adhered in large numbers to the same tissue sites in the neonatal brain sections as did the purified fimbriae, whereas the non-fimbriated host strain and a recombinant strain expressing P fimbriae did not adhere to brain tissues. The results suggest that adhesion of S-fimbriated bacteria to the binding sites observed in the neonatal brain has a pathogenetic role during bacterial invasion from circulation into the cerebrospinal fluid.

Animals↗
PubMed1987

Isolation and characterization of coliphage omega 18A specific for Escherichia coli O18ac strains.

The bacteriophage omega 18A, specific for Escherichia coli O18ac strains, was isolated from sewage. The results of host range and conjugation experiments showed that the sensitivity of bacteria to the phage is associated with the presence of O18ac antigens. With some of the O18 strains the phage omega 18A produces clear lysis on bacterial lawns only when applied at a high multiplicity and moreover the phage does not multiply. With the help of the phage omega 18A, E. coli O18ac strains could be divided into two serologically distinct subgroups called O18A and O18A1. E. coli strains belonging to the subgroup O18A are sensitive to phage omega 18A whereas bacteria of subgroup A1 are resistant.

Antigens, Bacterial↗
PubMed1987

Isolation and characterization of the alpha-sialyl-beta-2,3-galactosyl-specific adhesin from fimbriated Escherichia coli.

The alpha-sialyl-beta-2,3-galactosyl-specific adhesin (S adhesin) was isolated from cells of a recombinant Escherichia coli K-12 strain expressing the S-fimbrial adhesin complex. A crude cell extract was partially dissociated into fimbriae and an adhesin-enriched fraction by heating to 70 degrees C. From the latter, adhesin was purified to apparent homogeneity (by fast protein liquid chromatography, immunoblot, and NaDodSO4/PAGE) by differential ammonium sulfate precipitation, dissociation in 8 M guanidine hydrochloride, and high-resolution anion-exchange chromatography in 8 M urea. The purified adhesin formed an aggregate of Mr approximately 10(6) that was made up of one type of 12-kDa polypeptide (fimbrillin is 16.5 kDa). It had pI value of 4.7 (fimbriae has a pI value of 6). Adhesin and fimbrillin had different amino acid compositions. The purified adhesins agglutinated human and bovine erythrocytes with the same specificity as the whole bacteria; purified fimbriae were not adhesive. Monoclonal anti-adhesin and anti-fimbriae antibodies were obtained. Monoclonal anti-adhesin, but none of the anti-fimbriae, antibodies inhibited the agglutination of erythrocytes. The anti-adhesive antibodies were used in immuno-gold electron microscopy to localize adhesin exclusively on the fimbriae, with a possible preference to their tips.

Adhesins, Escherichia coli↗
PubMed1987

Comparison of the genetic determinant coding for the S-fimbrial adhesin (sfa) of Escherichia coli to other chromosomally encoded fimbrial determinants.

DNA probes specific for different regions of the S-fimbrial adhesin (sfa) determinant were constructed and hybridized with DNA sequences coding for P (F8 and F13), mannose-sensitive hemagglutinating type 1 (F1A), and F1C fimbriae. While the sfa and F1C DNA determinants exhibited homology along their entire lengths, the P-fimbrial and type 1-fimbrial determinants exhibited homology to regions of the sfa cluster responsible for the control of transcription and, to a minor extent, to regions coding for proteins involved in biogenesis and/or adhesion of the fimbriae and for the N-terminal part of the fimbrillin subunit.

Adhesins, Escherichia coli↗
PubMed1986

Influence of cloned Escherichia coli hemolysin genes, S-fimbriae and serum resistance on pathogenicity in different animal models.

The virulence of the uropathogenic E. coli strain 536 (O6:K15:H31) which produces the S-fimbrial adhesin (Sfa+), is serum-resistant (Sre+) and hemolytic (Hly+) and its derivatives were assessed in five different animal models. Cloned hemolysin (hly) determinants from the chromosomes of O6, O18 and O75 E. coli strains and from the plasmid pHly152 were introduced into the spontaneous Sfa-, Sre-, Hly- mutant 536-21 and its Sfa+, Sre+, Hly- variant 536-31. As already demonstrated for the 536-21 strains (Infect. Immun. 42: 57-63) the O18-hly determinant but not the plasmid-encoded hly determinant of pHly152 transformed into 536-31 contribute to lethality in a mouse peritonitis model. Similar results were obtained with both Hly- host strains and their Hly+ transformants in a chicken embryo test and in a mouse nephropathogenicity assay in which the renal bacterial counts were measured 15 min to 8 hours after i.v. infection. S-fimbriae and serum resistance had only a marginal influence in these three in vivo systems. In contrast all three factors, S-fimbriae, serum resistance and hemolysin, were necessary for full virulence in a respiratory mouse infection assay. In a subcutaneously-induced sepsis model in the mouse restoration of S-fimbriae and serum resistance and separately chromosomally-encoded hemolysis increased virulence to a level comparable to that of the parental 536 strain.

Animals↗
PubMed1986

Characterization of Escherichia coli wild-type strains by means of agglutination with antisera raised against cloned P-, S-, and MS-fimbriae antigens, hemagglutination, serotyping and hemolysin production.

E. coli strains isolated from patients with urinary tract infections (UTI) very often possess mannose-sensitive (MS) and mannose-resistant (MR) adherence factors (fimbriae). According to their receptor specificity the mannose-resistant adhesins can be divided into several types, P, S, M and X. We have cloned the determinants of three groups of UTI E. coli adhesins, MS, P and S, and prepared specific antisera against the fimbriae antigens. 189 hemagglutination (HA+)-positive strains, 96 fecal isolates and 93 strains isolated from UTI have been tested with these specific antisera and further characterized by receptor specific HA, HA patterns and further of the "common O serogroups" 01, 02, 04, 06, 07, 08, 018, 025, 075, most prevalent in UTI, and hemolysin production. 68 (73%) of the UTI strains and 50 (52%) of the fecal isolates showed P-receptor specificity; 16 (17%) of the uropathogenic bacteria and 33 (34%) of the fecal strains exhibited S, M or X-fimbriae antigens. 24% of the P-hemagglutinating (P+) strains reacted with P (F8)-specific antiserum. In contrast, more than three quarter of the S+-strains were agglutinated by S-specific antiserum. HA-pattern VI and 018 antigen were found to be associated with P-fimbriae strains, whereas HA-pattern V and VII and the O antigens 02 (M-type), 06 and 018 (S-type) occurred most frequently in P--strains. A high percentage of P-fimbriated strains showed mannose-sensitive hemagglutination and hemolysin production.

Adhesiveness↗
PubMed1986

Binding of Escherichia coli S fimbriae to human kidney epithelium.

Purified S fimbriae and an Escherichia coli strain carrying the recombinant plasmid pANN801-4 that encodes S fimbriae were tested for adhesion to frozen sections of human kidney. The fimbriae and the bacteria bound to the same tissue domains, and in both cases the binding was specifically inhibited by the receptor analog of S fimbria, sialyl(alpha 2-3)lactose. S fimbriae bound specifically to the epithelial elements in the kidneys; to the epithelial cells of proximal and distal tubules as well as of the collecting ducts and to the visceral and parietal glomerular epithelium. In addition, they bound to the vascular endothelium of glomeruli and of the renal interstitium. No binding to connective tissue elements was observed. The results suggest that the biological function of S fimbriae is to mediate the adhesion of E. coli to human epithelial and vascular endothelial cells.

Adenocarcinoma↗
PubMed1986

Analysis of the genetic determinants coding for the S-fimbrial adhesin (sfa) in different Escherichia coli strains causing meningitis or urinary tract infections.

Recently we have described the molecular cloning of the genetic determinant coding for the S-fimbrial adhesin (Sfa), a sialic acid-recognizing pilus frequently found among extraintestinal Escherichia coli isolates. Fimbriae from the resulting Sfa+ E. coli K-12 clone were isolated, and an Sfa-specific antiserum was prepared. Western blots indicate that S fimbriae isolated from different uropathogenic and meningitis-associated E. coli strains, including O83:K1 isolates, were serologically related. The Sfa-specific antibodies did not cross-react with P fimbriae, but did cross-react with F1C fimbriae. Furthermore the sfa+ recombinant DNAs and some cloned sfa-flanking regions were used as probes in Southern experiments. Chromosomal DNAs isolated from O18:K1 and O83:K1 meningitis strains with and without S fimbriae and from uropathogenic O6:K+ strains were hybridized against these sfa-specific probes. Only one copy of the sfa determinant was identified on the chromosome of these strains. No sfa-specific sequences were observed on the chromosome of E. coli K-12 strains and an O7:K1 isolate. With the exception of small alterations in the sfa-coding region the genetic determinants for S fimbriae were identical in uropathogenic O6:K+ and meningitis O18:K1 and O83:K1 strains. The sfa determinant was also detected on the chromosome of K1 isolates with an Sfa-negative phenotype, and specific cross-hybridization signals were visible after blotting against F1C-specific DNA. In addition homology among the different strains was observed in the sfa-flanking regions.

Adhesins, Escherichia coli↗
PubMed1986

Contribution of cloned virulence factors from uropathogenic Escherichia coli strains to nephropathogenicity in an experimental rat pyelonephritis model.

Escherichia coli 536 (O6:K15:H31), which was isolated from a case of urinary tract infection, determines high nephropathogenicity in a rat pyelonephritis system as measured by renal bacterial counts 7 days after infection. The loss of S fimbrial adhesin formation (Sfa-) (mannose-resistant hemagglutination [Mrh-] and fimbria production [Fim-]), serum resistance (Sre-), and hemolysin production (Hly-) in the mutant 536-21 led to a dramatic reduction of bacterial counts from almost 10(5) to only 40 cells per g of kidney. The reintroduction of the cloned S fimbrial adhesin determinant (sfa) increases the virulence of the avirulent mutant strain by a factor of 20; almost the same effect was observed after restoration of serum resistance by integration of an sfa+ recombinant cosmid into the chromosome. Additional reintroduction of the Hly+ phenotype by transformation of two hly determinants increased the virulence of the strains. Hemolysin production determined increased renal elimination of leukocytes and erythrocytes. Thus all three determinants investigated, S fimbriae, serum resistance, and hemolysin, contribute to the multifactorial phenomenon of E. coli nephropathogenicity.

Adhesins, Escherichia coli↗
PubMed1986

Role of Escherichia coli alpha-hemolysin and bacterial adherence in infection: requirement for release of inflammatory mediators from granulocytes and mast cells.

We investigated the role of bacterial mannose-resistant fimbriation of S fimbriae (Fim), mannose-resistant hemagglutination (S-Mrh), and hemolysin (Hly) production by an Escherichia coli parent and genetically cloned strains as regards their effect on histamine release from rat mast cells and generation of the chemiluminescence response, leukotriene, and enzyme release from human polymorphonuclear granulocytes. These mediators are involved in the induction of inflammatory disease processes and lead, e.g., to the enhancement of vascular permeability, chemotaxis, aggregation of granulocytes (leukotriene B4), lysosomal enzyme release, and smooth-muscle contraction (leukotrienes C4, D4, and E4). The content of azurophilic and specific granules in polymorphonuclear granulocytes consists of highly reactive enzymes which amplify inflammatory reactions. Washed bacteria (E. coli 764 Hly+/-, E. coli 21085 Hly+/- Fim+/- Mrh+/-), as well as their culture supernatants, were analyzed at various times during their growth cycle. No differences exist between parent and cloned or mutant strains with respect to their outer membrane proteins and lipopolysaccharide pattern. Washed bacteria [E. coli 764 and 21085(pANN202-312)] which produced hemolysin, unlike Hly- strains, induced high levels of histamine release from rat mast cells and led to a significant chemiluminescence response and enzyme and leukotriene release from human polymorphonuclear granulocytes. Bacterial culture supernatants from Hly+ and secreting strains showed similar results with the exception of E. coli 21085(pANN202-312), which is a hemolysin-producing but not a secretory strain. Our data suggest a potent role for hemolysin as a stimulus for noncytotoxic mediator release from various cells. Furthermore, we showed that the presence of Fim and S Mrh potentiates mediator release. The simultaneous presence of Mrh and Fim [E. coli 535/21(pANN801-4)] increased mediator release compared with Mrh+ Fim- strains [E. coli 536/21(pANN801-1)]. E. coli 536/21 (Msh- Mrh- Fim- Hly-) did not induce mediator release.

Arachidonic Acid↗
PubMed1986

Large, unstable inserts in the chromosome affect virulence properties of uropathogenic Escherichia coli O6 strain 536.

The hemolytic, uropathogenic Escherichia coli 536 (O6:K15:H31) contains two inserts in its chromosome (insert I and insert II), both of which carried hly genes, were rather unstable, and were deleted spontaneously with a frequency of 10(-3) to 10(-4). These inserts were not found in the chromosome of two nonhemolytic E. coli strains, whereas the chromosomal sequences adjacent to these inserts appeared to be again homologous in the uropathogenic and two other E. coli strains. Insert I was 75 kilobases in size and was flanked at both ends by 16 base pairs (bp) (TTCGACTCCTGTGATC) which were arranged in direct orientation. For insert I it was demonstrated that deletion occurred by recombination between the two 16-bp flanking sequences, since mutants lacking this insert still carried a single copy of the 16-bp sequence in the chromosome. Both inserts contained a functional hemolysin determinant. However, the loss of the inserts not only affected the hemolytic phenotype but led to a considerable reduction in serum resistance and the loss of mannose-resistant hemagglutination, caused by the presence of S-type fimbriae (sfa). It is shown that the Sfa-negative phenotype is due to a block in transcription of the sfa genes. Mutants of strain 536 which lacked both inserts were entirely avirulent when tested in several animal model systems.

Adhesins, Escherichia coli↗
PubMed1985

Analysis of the flanking regions from different haemolysin determinants of Escherichia coli.

The haemolysin (hly) determinant of the plasmid pHly152 contains an IS2 element at 469 bp upstream of the hlyC gene. The sequence at the other (right-hand) end (RS) also shows multiple hybridization with the plasmid pHly152 and the chromosome of some Escherichia coli strains but the nucleotide sequence of this region does not reveal the typical properties of an IS element. Similar arrangements in the regions flanking the hly determinant are also found on various Hly plasmids from uropathogenic E. coli strains. Chromosomal hly determinants lack both flanking sequences (IS2 and RS) in the immediate vicinity of the hly genes. The sequences immediately upstream of the hlyC gene have been determined from several chromosomal hly determinants and compared with the corresponding sequence of the hly determinant of the plasmid pHly152. We show that these sequences, which contain one promoter (left promoter, phlyL) in all hly determinants tested, vary considerably although common sequence elements can still be identified. In contrast, only relatively few nucleotide exchanges have been detected in the adjacent structural hlyC genes. The A + T content of the 200 bp sequence upstream of hlyC is very high (72 mol% A + T) but even the structural hly genes show a considerably higher A + T content (about 60 mol%) than the E. coli chromosome on average (50 mol% A + T) suggesting that the hly determinant may not have originated in E. coli.

Amino Acid Sequence↗
PubMed1985

Cloning and characterization of genes involved in production of mannose-resistant, neuraminidase-susceptible (X) fimbriae from a uropathogenic O6:K15:H31 Escherichia coli strain.

The uropathogenic Escherichia coli strain 536 (O6:K15:H31) exhibits a mannose-resistant hemagglutination phenotype (Mrh) with bovine erythrocytes and delayed Mrh with human and guinea pig erythrocytes. Neuraminidase treatment of the erythrocytes abolishes mannose resistant hemagglutination, which is typical for X fimbriae. E. coli strain 536 synthesizes two different fimbriae (Fim phenotype) protein subunits, 16.5 and 22 kilodaltons in size. In addition the strain shows mannose-sensitive hemagglutination and common type I (F1) fimbriae. The cosmid clone E. coli K-12(pANN801) and another nine independently isolated Mrh+ cosmid clones derived from a cosmid gene bank of strain 536 express the 16.5-kilodalton protein band, but not the 22-kilodalton protein, indicating an association of the Mrh+ property with the "16.5-kilodalton fimbriae." All cosmid clones were fimbriated, and they reacted with antiserum produced against Mrh+ fimbriae of the E. coli strain HB101(pANN801) and lacked mannose-sensitive hemagglutination (F1) fimbriae. From the Mrh fim cosmid DNA pANN801, several subclones coding for hemagglutination and X fimbriae were constructed. Subclones that express both hemagglutination and fimbriae and subclones that only code for the hemagglutination antigen were isolated; subclones that only produce fimbriae were not detected. By transposon Tn5 mutagenesis we demonstrated that about 6.5 kilobases of DNA is required for the Mrh+ Fim+ phenotype, and the 1.5- to 2-kilobase DNA region coding for the structural protein of the fimbriae has been mapped adjacent to the region responsible for the Mrh+ phenotype. Two different regions can thus be distinguished in the adhesion determinant, one coding for hemagglutination and the other coding for fimbria formation. Transformation of plasmid DNA from these subclones into a Mrh- Fim- mutant of E. coli 536 and into a galE (rough) strain of Salmonella typhimurium yielded transformants that expressed both hemagglutination and fimbria production.

Animals↗